Preservation of semen



United States Patent 3,472,735 PRESERVATION 0F SEMEN Yoshimasa Nishikawa, Ashiya, and Toshio Hayami, Tokyo, Japan, assignors to Takeda Chemical Industries, Ltd., Higashi-ku, Osaka, Japan No Drawing. Filed Apr. 25, 1967, Ser. No. 633,366 Claims priority, application Japan, Apr. 28, 1966, 41/27,264; June 8, 1966, 41/37,274 Int. Cl. C12k 9/00; A61k 17/06 US. Cl. 1951.8 33 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a method for heightening and maintaining the livability and motility of the spermatozoa in the semen preparation for artificial insemination of livestock and also to a stabilized semen preparation for artificial insemination of livestock.

In recent years, artificial insemination has widely been accepted in livestock reproduction such as cattle, goat and sheep. The object of which of course is not only to reproduce farm animals efiiciently but also to make full use of selective semen from proven sires for the genetic improvement of breed. For effecting artificial insemination, semen collected from the male animal is at first stored outside the body and then inseminated to the female animal selected optionally. Therefore, the reasonable operation of the artificial insemination depends fully upon the satis factory storage of semen for a long time outside the body.

In general, the animal semen for artificial insemination is stored after it is diluted with a suitable semen diluter, and the kind of the diluter and the temperature for the storage have close connection with the livability and motility of the spermatozoa in the diluted semen, and thus with the fertilizing capacity thereof.

For the storage of the diluted semen, the metbolism of the spermatozoa must be appropriately suppressed. Lowering the storage temperature is generally adopted for this purpose. In this viewpoint, the hitherto-employed processes for storing semen for artificial insemination can be divided into two groups i.e. so-called liquid semen method and so-called deep freezing method. The liquid semen method consists in diluting semen collected from male animals to about to 20 times of its original volume with a semen diluter and subsequently keeping the diluted semen at a low temperature of about 4 C. until actual use thereof.

The deep freezing method comprises diluting semen collected from male animals to about 3 to times of its original volume with a semen diluter (the primary dilution), cooling the resultant diluted semen at about 4 C. gradually for about 1 hour, further diluting the diluted semen to about 2 times of the volume with the semen diluter supplemented with glycerin as an antifreezing agent (secondary dilution), keeping the diluted semen at about 4 C. for about 6 to 18 hours in order to permeate glycerin into the spermatozoan cells (glycerin equillibrium), then freezing and storing at a ultra-low temperature of about -80 C. or of about 196 C., and thawing the frozen semen at a temperature of about 4 C. or about 37 C. before the use of the semen.

The liquid semen method, however, entails drawbacks that the livability and motility of the spermatozoa in the diluted semen lowers with the lapse of the storage time since the metabolism of spermatozoa cannot be sufficiently inhibited, and therefore, the artificial insemination with the use of the diluted semen stored for relatively long period beyond five days results in low conception rate. Similarly the deep freezing method entails drawbacks that the livability and motility of spermatozoa in the thawed semen obtained by thawing the frozen semen lowers with the lapse of time although the livability and motility of the spermatozoa in the frozen semen are kept at rather stable conditions throughout the period of freezing storage.

Thus, it has been one of the most important desirata in the field of stockbreeding to establish a method for keeping spermatozoa in the diluted semen and the thawed semen from the frozen semen viable for a long time.

It is an object of the present invention to provide a method for heightening and maintaining the livability and motility of spermatozoa in a semen preparation for artificial insemination of livestock. Another object of the present invention is to provide a stabilized semen preparation suitable for a long storage of the semen. Other objects will be apparent from the detailed description of the present invention hereinafter provided.

In accordance with one embodiment of the present invention, it is now found that the livability and motility of the spermatozoa of livestock, especially of ruminant livestock such as cattle, goat, sheep, in the semen preparation e.g. the diluted semen and the thawed semen from the frozen semen can be heightened and maintained so that the fertilizing capacity of the spermatozoa can be heightened and maintained for an extended period of time by the presence of a small amount of a certain thiol-type thiamine derivatives.

The thiol-type thiamine derivatives to be contained in the semen preparation includes unsymmetrical thiamine organic disulfide derivatives, S-acyl-thiamine derivatives and bisthiamine-disulfide derivatives.

As the unsymmetrical thiamine organic disulfide derivatives, there may be enumerated the following compounds.

UNSYMMETRIOAL THIAMINE ORGANIC DISULFIDE As the S-acyl-thiamine derivatives there may be enumerated the following compounds.

S-ACYL-THIAMINE! DERIVATIVES As the bisthiamine-disulfide derivatives there may be enumerated the following compounds.

BISTHIAMINE-DISULFIDE DE RIVATIVES NH; (in s- I I -C 2114 O -R or. K

Compounds R Thiamine-disulfide O-benzoyIthlamlne-dlsulfide The said thiol-type thiamine derivatives may be employed as free compounds or corresponding salts with a suitable acid such as hydrochloric acid and nitric acid. In the method of the present invention one compound or a mixture of more than one of these thiol-type thiamine derivatives is added to the semen preparation for artificial insemination. From the viewpoint of effect of heightening and maintaining the livability and motility of the sperma tozoa in the semen preparation, the thiol-type thiamine is generally used in such an amount as about 0.001 to about 0.1 weight percent preferably about 0.006 to about 0.01 weight percent relative to the total volume of the semen preparation.

The semen preparation to be incorporated with the thiol-type thiamine derivative may be any of the conventional semen preparations for artificial insemination of livestock. That is to say, according to the method of the present invention, either the diluted semen in the abovementioned liquid semen method or the thawed semen obtained by thawing the frozen semen in the above-mentioned deep freezing method can be heightened and maintained high in the livability and motility of the spermatozoa. And such effect of heightening and maintaining the livability and motility of the spermatozoa can be attained by adding a thiol-type thiamine derivative to any conventional semen preparation before the use. In a case of the diluted semen in the liquid semen method, the thiol-type thiamine derivative may be added to the semen diluter with which the semen is diluted, to the diluted semen just after the dilution, or to the diluted semen under the storage at about 4 C. In a case of the thawed semen in the deep freezing method, the thiol-type thiamine derivative may be added to the semen in any of the steps of the second dilution, of the glycerin equilibrium and of the storage at about 4 C. after thawing. Most practically, the object of the present invention can be attained by diluting the semen with a semen diluter containing the thiol-type thiamine derivative, which is prepared by adding the derivative to any of hitherto known diluters. In this case, the concentration of the thiol-type thiamine in the semen diluter is preferably about 0.001 to 0.1 weight percent, most preferably about 0.006 to 0.01 weight percent relative to the volume of the diluter.

As the semen diluter to be employed in the present invention may be any of the ordinary diluter known in the current art. In general, the diluters may comprise, as a basal component, egg yolk, egg product such as lecithin, milk or milk product such as defatted milk or caesin, and additionally may contain inorganic and organic buffering salts, energy sources such as fructose and amino acid, all of which are familiar in the veterinary and animal husbandry art, and are commonly chosen depending upon the factors such as kind of the livestock. As the examples of such semen diluter there may be enumerated an egg yolk diluter such as egg yolk citrate buffer solution, egg yolk phosphate buffer solution, a milk diluter which is prepared by heating whole milk or skim milk.

Thus prepared semen preparation containing the thioltype thiamine derivative is very high in the livability and motility of the spermatozoa therein and therefore the fertilizing capacity of the spermatozoa in the semen preparation can persist after many days of storage.

The following examples are merely intended to illustrate presently preferred embodiments of the methods and compositions of the present invention and not to restrict the scope of the latter.

Throughout the present specification as well as in the following examples, the abbreviations ml. and C. respectively refer to milliliters and degrees centigrade, and percentages are weight/volume unless otherwise described:

In the following examples, the livability and the motility of spermatozoa is shown by the following references:

++'ISpermatozoa with very actively progressive motion.

++fiSpermatozoa with actively progressive motion.

+Spermatozoa' with progressive motion.

:Rotative or pendulum-like motile spermatozoa.

+++-++Spermatozoa with the middle motion between said and ++-+Spermatozoa with the middle motion between said++ and +-:Spermatozoa with the middle motion between said l and i.

Percentages which are described in front of said references mean percentage of spermatozoa with such motion to all spermatozoa in the semen preparation. For example,

means that percentage of spermatozoa with very actively progressive motion and percentage of spermatozoa with actively progressive motion to all spermatozoa in the semen preparation are and 5 respectively.

3,472,735 5 6 And the livability indexes of spermatozoa in the semen 0.0020000% of thiamine propyl disulfide. Each of the preparation are shown in the numerals calculated from diluted semen samples is cooled to 4 C and stored at the following formula. even temperature for 7 days. The livability and motility of the spermatozoa in one drop of each of the diluted Li bflit indeFW semen samples is examined microscopically at about 4' 100 5 C. The results are shown in the following Table 1.

TABLE 1 Concentra- Storage period (days) tion 01 thia- Bulls mine propyl disulfide in Immethe semen diately 1 2 3 4 5 6 7 diiuter alter the (percent) dilution 0 0078125 s0+++ 50+++v 15+++ fi5+++ 55+++ 60+++-++ 50+++-++ 45++ A 5+ 5+ 5+ 5+ 5+ 5+ 5+ 15+ MO t on 70+++ 7o+++ 7o+++ 55+++ 50+++ 40+++-++ 45++ 30++-+ 10+++ 7 0+++ 0+++ ao++-+ 0 (Control) 30+-+ 20+ at =Motile but unnumbered.

wherein Example 2 a.--Percentage of to all spermatozoa in th semen Bovine semen collected from one bull is diluted 10-fold preparation at the body temperature w1th an egg yolk citrate buffer b Percentage of solution containing 2.4% of sodium citrate and 20% of c percemage of fresh yolk. The diluted semen is stored at 4 C. After d Pel centage of two days storage, thiamine tetrahydrofurfuryl disulfide,

thiamine tetrahydrofurfuryl disulfide hydrochloride, thiamine propyl disulfide and thiamine propyl disulfide hydrochloride is added to the diluted semen in a final concentration of 1%, 0.5% or 0.125%.

Example 1 Bovine semen collected from 2 bulls is diluted 10-fold at the body temperature with an egg yolk citrate butler solution containing 2.4% of sodium citrate and 20% of fresh yolk and with the semen diluters of the present invention prepared by supplementing the said egg citrate buffer solution with 0.015250%, 0.007s12s% or The diluted semen is further stored at 4 C. for 5 days. Throughout the period of the storage, the livability and motility of spermatozoa in the diluted semen is examined microscopically. The results are shown in the following Table 2.

TABLE2 Storage period (days) 2 Thio-type Coneeu- Immethiamine tration diately derivatives (percent) after the 1 Before Imme- 3 4 5 6 7 dilution the diately addition after the addition TTFD 0.5000 85+++ 5+ 20+-+ 0 70+++ 75+++ 70+++ 55 40 35 0052: s5+++ s0+++ so+++ TABLE 2-Continued Storage period (days) 2 'Ihlo-type Concen- Immethiamine tration diately derivatives (percent) after the 1 Before Imme- 3 4 5 6 7 dilution the diately addition after the addition 7 1.0000 85+++ s0+++ 85+++ et' 0 TTFD 70+++ hydro- 0.5000 85+++ 80+++ 80+++ 0 chloride. 5+

7 TPD 0.5000 85+++ 80+++ 5+ 85+++ 10;}; 0

70+++ 1.0000 85+++ 80+++ 5+ 85+++ 0 'lPD 10 so++ hydro- 0.5000 85+++ 80+++ 10+-i 0 chloride. 5+ 5+ 7 7 40++ 40+ Control A..- 0 85+++ 80+++ 70 70 70 50 40 ControlB-n o 85+++ 80+++ 20+-=t TTFD =thiamine tetrahydroiuriuryl disulfide.

Example 3 TPD =thiamine propyl disulfide:

mine propyl disulfide and thiamine propyl disulfide hydrochloride are added to the diluted semen in concentra tions described in the following Table 3. The diluted semen is further stored at 4 C. for 3 days. Throughout the period of the storage, the livability and motility of spermatozoa in the diluted semen is examined microscopically. The results are shown in the following Table 3.

TABLE 3 Storage period (days) Concentration of test 4 Thiol-type compounds in derivatives the diluted Immedisemen ateiy after 1 2 3 Before the Immediately 5 6 7 (percent) the dilution addition after the of test addition compounds of test compounds 7 65 65 TTFD 0.031250 85+++ 75+++ 65+++ 9 10 TABLE 3Con tlnued Storage period (days) Concentration of test 4 Thiol-type compounds in derivatives the diluted Immedisemen ately after 1 2 3 Before the Immediately 5 6 7 (percent) the dilution addition after the of test addition compounds 01 test compounds 60+++ 65+ 75 0.062500 85+++ 85+++ 75+++ 65+++ 75+++ 70+++ T' l lg 5+ 5+ 5+ y rochloride. 60 65 65 60 7 TPD 0.031250 85+++ 85+++ 05+++ 0+++ 70+++ TPD 60+ 60 70 6 hydro- 0.031250 85+++ 85+++ 75+++ 65+++ 5+++ 60+++ chloride. 5+ 10+ 5+ 10+ 5+ 60 ControlAn 0 50+++ 50++ 50++ 60++ 60 50 50 Conn-01B" 0 85+++ 85+++ 7 50++ 1 'ITFD =thiamine tetrahydroiurfuryl disulfide.

at the body temperature with a physiological saline solution and the mixture is subjected to centrifugation to get rid of seminal plasma. After decanting the supernatant, sedimenting spermatozoa is resuspended into an egg yolk citrate buffer solution containing 2.4% of sodium citrate and 20% of fresh yolk in the same volume as the original semen.

The resulting semen is diluted 10-fo1d at the body B TPD =thiamine propyl disulfide.

the same composition as described above. The diluted is stored at 4 C. After two days storage, thiamine tetrahydrofurfuryl disulfide, thiamine tetrahydrofurfuryl disulfide hydrochloride, thiamine propyl disulfide and thiamine propyl disulfide hydrochloride are added to the diluted semen in concentrations described in the following Table 4. The dilute semen is further stored at 4 C. for 5 days. Throughout the period of the storage, the livability and motility of the spermatozoa in the diluted semen is examined microscopically. The results are shown temperature with an egg yolk citrate buffer solution of 55 in the following Table 4.

TABLE 4 Storage period (days) Coneem tration 2 'lhiol-type of test thiamine compounds derivan the Immetives diluted Before the dlatel semen 1 addition after the 3 4 5 6 7 (percent) of test addition compounds of test compounds 5 5 7 TTFD L... 0. 5000 0 0 0 0 0 sequently removing therefrom milk fat by centrifuganon for 30 minutes at 3000 r.p.m. To the diluted semen is added thiamine propyl disulfide hydrochloride in concentrations of 0.00313% and 0.00156%. Each of the diluted semen is stored at 4 C. for 7 days. The livability and motility of spermatozoa in the diluted semen is examined microscopically. The results are shown in Table 5.

arter the manner described in Example 5. The diluted semen is stored at 4 C. After two days storage, thiamine propyl disulfide hydrochloride is added to the diluted solution in concentration of 0.01250%, 0.00625 0.00313% and 0.=00156%. Each of diluted semen samples is further stored at 4 C. for 5 days. The livability and motility of spermatozoa in the respective diluted semen samples is shown in the following Table 6.

TABLE 5 Concen- Storage period (days) tration of thiamine propyl 2 Bulls disulfide hydrochloride 1 3 4 5 6 7 in the diluted Before the Immediately semen addition after the (percent) addition on m 5+ 5+ 5+ 5+ 5+ 5+ 10+ 10+ 5* A 0 00313 10+++ 60+++ 55+++ 55+++ 1o+++ 50+++ 45+++-++ 20++-+ 2o++-+ 0 C t 1) 60+++ 55+++ 5u+++ 50+++ 45+++-++ so++ 20++-+ 20++-+ 20++- 0 00313 5o+++ 55+++ 50+++ 55+++ 55+++ 55+++-++ a5++ 20++-+ 20++-+ B 5+ 5+ 10+ 5+ 5+ 10+ 10+ 10+ 5+ Example 6 Example 7 Bovine semen collected from 3 bull's is diluted lO-fold 45 at the body temperature with a milk dilutor prepared Bovine semen collected from 2 bulls is diluted 10-f0ld at the body temperature with a milk diluter prepared TABLE 7 Concen- Storage period (days) tration of thiamine p w 4 Bulls disulfided hygglgtlilgg e 0 1 2 3 Before the Immediately 5 6 7 the diluted addition after the semen addition (percent) 1 7 4 4 20++-+ 10+ 0 (Comm 6+ 5+ 10+ 10+ 5+ 5+ 5+ 10d: 10*

7 0+++ 4 0 (Control) 5+ 5+ 10+ 5+ 5+ 5+ 5+ 5+ 5+ 15 after the manner described in Example 5. The diluted semen is stored at 4 C. After four days storage, thiamine of the livability index of the respective thawed semen samples is shown in the following Table 8.

TABLE 8 Livability index Concentration of test Before the refrigeration After thawing Bulls Thiol-type thiamine derivatives compfilund 111 Q thawed Before Just after Just after 4 hrs. 12 hrs. 24 hrs. semen the addition the addition thawing after after after of test oi test thawing thawing thawing compounds compounds 0.01136 75. 0 75. 0 67. 5 67. 5 62. 5 48. 3 Thiamine propyl disulfide 0. 01136 75. 0 75. 0 62. 5 62. 5 62. 5 48. 3 Thiamine propyl disulfide hydrochloride..

Control 0 75. 0 57. 5 52. 9 40. 0 33. 3

0. 001136 80. 0 80. 0 62. 5 67. 5 67. 5 62. 5 Thiamine propyldisulfide 0. 01136 80. 0 80. 0 62. 5 67. 5 67. 5 57. 5 Thiamine propyl disulfide hydrochloride Control 0 80. 0 57. 5 52. 9 52. 9 30. 4

propyl disulfide hydrochloride is added to the diluted Example 9 solution in concentrations of 0.00313% and 0.00156%. Each of the diluted semen samples is further stored at 4 C. for 3 days. The livability and motility of spermatozoa in the respective diluted semen samples is shown in Table 7.

Example 8 Bovine semen collected from 2 bulls is diluted S-fold at the body temperature with an egg yolk citrate buffer solution containing 2.4% of sodium citrate and 20% of fresh yolk, and the resulting diluted semen is cooled to 4 C. gradually for 1 hour. The diluted semen is further diluted 2-fold at 4 C. with an egg yolk citrate buffer solution containing 2.4% of sodium citrate, 20% of fresh yolk and 14.0% of glycerin. To the resultant diluted semen are added thiamine propyl disulfide and thiamine propyl disulfide hydrochloride in concentrations described in the following Table 8. And then the diluted semen is kept standing at 4 C. for 10 hours. 1 ml. each of the diluted semen samples is put into ampoules of 2 ml. capacity and the ampoules are closed. After being frozen and stored in a liquid-nitrogen refrigerator at -196 C. for 3 days, each of the ampouled samples is thawed at 4 C. and stored at even temperature for 24 hours. The change Goat semen collected from 2 sires is diluted 5-fold at the body temperature with a physiological saline solution and the resultant mixture is subjected to centrifugation to get rid of seminal plasma. After decanting the supernatant, sedimenting spermatozoa is resuspended into an egg yolk citrate buffer solution containing 2.4% of sodium citrate and 20% of fresh yolk in the same volume as the original semen. The resultant semen is diluted S-fold at the body temperature with an egg yolk citrate buffer solution of the same composition as described above. After being cooled to 4 C. gradually for 1 hour, the diluted semen is further diluted 2-fold with an egg yolk citrate buffer solution containing 2.4% of sodium citrate and 20% of fresh yolk, 14% of glycerin. To the resultant diluted semen are added thiamine propyl disulfide and thiamine propyl disulfide hydrochloride in concentrations described in the following Table 9. And then the diluted semen is kept standing at 4 C. for 10 hours. 1 ml. each of the diluted semen samples is put into ampoules of 2 ml. capacity and the ampoules are closed. After being frozen and stored in Dry Ice refrigerator at -79 C. for 3 days, each of the ampouled samples are thawed at 4 C. and stored at even temperature for 24 hours. The change of livability index of the respective thawed semen samples is shown in the following Table 9.

TABLE 9 Livahility index Concentration of test Before the refrigeration After thawing Bulls Thiol-type thiamlne denvatives co ptrilund e thawed Before Just after Just after 4 hrs. 12 hrs. 24 hrs. semen the addition the addition thawing after after after of test oi test thawing thawing thawing compounds compounds 0. 00568 80. 0 80. 0 39. 2 52. 4 62. 5 57. 5 Thiamine propyl disulfide 0. 00568 80. 0 80. 0 43. 7 52. 5 57. 5 57. 5 Thiamine propyldisulflde hydrochloride.-.

Control 0 80. 0 80.0 31. 7 31. 7 31. 7 17. 5

Thiamine propyldisulflde 0- 00568 80. 0 80. 0 52. 9 52. 9 52. 9 44. 2

Thiamine propyldisulfide hydrochloride... 0.00568 80. 0 80.0 67.5 I 62. 5 62. 5 57. 5

Control 0 80. 0 80. 0 52. 9 52. 9 40. 0 26. 7

Example 10 Bovine semen collected from 2 bulls is diluted S-fold at the body temperature with an egg yolk citrate buffer solution containing 2.4% of sodium citrate, buffer yolk and 14% of glycerin. After being kept standing at 4 C. for 10 hours, the diluted semen samples is put solution containing 2.4% of sodium citrate and 20% of into p les a the ampoules are closed. \fter being fresh yolk, and the resulting diluted semen is cooled to 4 frozen and Stored In a hqwd-mtrogen refrlgerator at C. gradually for 1 hour. The diluted semen is further 196 C. for 3 days, each of the ampouled samples 15 diluted 2-fold at 4 C. with an egg yolk citrate buifer thawed at 4 C. To the thawed semen samples are added TABLE 10 Livability index Ctonteen- 0 o i tst After thawing -t thi ine derivatives com ounds Before Buns Thml ype am it? the the refrigthawed eration Just after Just after 4 hrs. 12 hrs. 24 hrs.

semen thawing after the after the after the after the addition addition addition addition A Thiamine propyldisulfide 0.01136 80.0 62.9 62.5 67.5 52.9 52.9

r1 Thiamme propyldjsulfide hydmcmo de 0.00568 80.0 52.9 57.6 62.5 62.5 67.5

Control 0 80.0 52.9 62.9 62.9 35.3 35.8

TABLE 10-Continued Livability index Concentration of tests After thawing Bulls Thiol-type thiamine derivatives compounds Before in the the refrigthawed eration Just after Just after 4 hrs. 12 hrs. 24 hrs.

semen thawing after the after the after the after the addition addition addition addition 0.01136 80. 0 62. 5 67. 5 72. 5 62. 5 62. 5 Thiamine propyl disulfide 0. 01136 80. 0 62. 5 72. 5 72. 5 67. 5 62. 5 Thiamine propyl disulfide hydrochloride Control 0 80.0 62. 5 62. 5 62.5 44. 2 40. 2

thiamine propyl disulfide and thiamine propyl disulfide hydrochloride in concentrations described in the Table 10. And then the thawed semen samples are further stored at 4 C. for 24 hours. The change of the livability index of the respective thawed semen samples is shown in the Table 10.

Example 11 same composition as described above. After being cooled to 4 C. gradually for 1 hour. The diluted semen is further diluted 2-fold with an egg yolk citrate buffer solution containing 2.4% of sodium citrate, 20% of fresh yolk and 14% of glycerin. After being kept standing at 4 C. for 11 hours, 1 ml. each of the diluted semen samples is put into ampoules and the ampoules are closed. After being frozen and stored in a liquid-nitrogen refrigerator at -196 C. for 3 days, each of the ampouled samples is thawed at 4 C. To the thawed semen samples are added thiamine propyl disulfide or thiamine propyl disulfied hydrochloride in concentrations described in the following Table 11, and then the thawed semen samples are stored at 4 C. for 24 hours. The change of the livability index of the respective thawed samples is shown in the Table 11.

TABLE 11 Livability index Concentration of tests compounds After thawing Bulls Thiol-type thiamine derivatives in the Before thawed the refrigsemen eration Just after Just 4 hrs. 12 hrs. 24 hrs.

thawing after the after the after the after the addition addition addition addition A Thiamine propyl disulfide 0. 00568 80. 0 27. 5 39. 5 39. 5 48. 3 48.3

Thiamine propyl disulfide hydrochloride 0.00568 80. 0 27. 5 39. 5 57. 5 62. 5 57. 5

Contro1 0 80.0 27. 5 27. 5 27. 5 27. 5 8. 3

TABLE 11Continued Livsbllity index, Concentration of tests compounds After thawing Bulls 'Ihiol-type thiamine derivatives in the Before thawed the refrlgsemen eration Just after Just 4 hrs. 12 hrs. 24 hrs.

thawing after the alter the after the after the addition addition addition addition Thiamine propyl disulfide 0. 00568 80. 0 27. 5 57. 5 57.5 57. 7 52. 9

Thiamine propyl disulfide hydrochloride 0. 00568 80. 0 27. 5 57. 5 62. 5 62. 5 57. 5

Control 0 80. 0 27. 5 48. 3 52. 9 40. 0 26. 7

Example 12 Bovine semen collected from 2 bulls is diluted -fold with anfigg yolk citrate buifer solution containing 2.4% of sodium citrate and of egg yolk and the diluted described in the following Table 12 in a concentration of 0.0125 0.0625 or 0.0313%. Each of the diluted semen is stored at 4 C. for 4 days. The livability and motility of the spermatozoa in the diluted semen is shown semen are mixed with the thioltype thiamine derivatives in the Table TABLE 12 Storage period (days) Concen- Bulls Thlol-type thiamine tration derivatives (percent) Just a after the 1 2 3 4 addition 8 8 Thiamine propyl disulfide hydrochloride 0. 00625 8 b 1 h m o h h t 0 00625 so+++ 75+++ 1 55+++ enzoy-t is no motion osp a e 5+ 10+ 5+ 5+ 5+ 0 Sm t h d m id 0 00625 80+++ 5+++ 4 ace ylthiamine y roe or e 5+ 5+ 5+ 5+ 5+ 0 00625 75+++ O,S-eyelocarbo thiamine hydrochloride 5+ 5+ 5+ 10+ 10+ TABLE 12-C0ntinued Storage period (days) Th1 it :51 1 5 5 B 3 re on Buns der i atives n6 (percent) Just after the 1 2 3 4 addition 0 so+++ so+++ 15+++ 1 8 8 7 TATD 0. 00625 50++ 65 60+++ 50 O-benzoyl-thigmiue-disulfide 0. 00625 Thiamine 2-hydr0xymethyl disulfide hydro 80+++ 80+++ 70+++ 65+++ 55+++-++ chloride 0. 00625 A 5+ 5+ 80+ 80 70 60+++ 0,5-dibenzoy1-thiamine 0.00625 80 80 70 60 50 0,S-dicarboothoxy thiamine hydrochloride 0. 00625 8 7 50+++-++ 35++ Control 0 80 0 0 0. 01250 8 +:H-' 8 Not observed m++ 80 80 80 6 Thiamine propyl dlsulfide hydrochloride 0. 00625 Not observed 6+++ 80 5 0.00313 7 Not observed 50+++ 80 0 0.01250 8 Not'observed 80 70 55 B s-benzoyl-thaimine O-mono-phosphate 0. 00625 Not observed 80 70 60 45 0.00313 Not observed 80 70 60 4 0. 01250 Not observed 5+++ 80 75 70 40 0,S-d1ecetyl-thiamine hydrochloride 0.00625 Not observed 80 70 60 0 0 00313 Not observed 4 TABLE 12-Continued Storage period (days) Concen- Bulls Thiol-type thiamine tration derivatives (percent) Just after the 1 2 3 4 addition 7 0. 01250 Not observed 80++ 75 70++ 40++ 0,S-eyclocerbo thiamine hydrochloride 0. 00625 Not observed 7 40+++-++ 0. 00313 Not observed B 10+ 10+ 0. 01250 Not observed 80+ 80 70+ 50+ TATD I 0. 00625 Not observed 4 0. 00313 Not observed 0. 01250 Not observed 8 O-benzoyltbiamine-disnlfide 0. 00625 Not observed 0. 00313 Not observed 80++ 80 "5 30++ 0.01250 I Not observed 8 7 Thiamine-2hydroxy-ethyl disuifide hydro- 0. 00625 N 01; observed chloride. 5+ 5+ 10+ 5+ 80++ 70 60 50 0. 00313 Not observed 8 7 60+++ Control 0 Not observed 1 TATD=thiamine (7-methoxycarbonyl-3-acetylthioheptyl) disulfide.

Example 13 Sheep semen collected from 2 sires is diluted 10-fold with an egg yolk cirate buifer solution containing 2.4% 5 of sodium citrate, 20% of fresh yolk and 0.00625% of- TABLE 13 Concentre- Storage period (days) tion of v test com- Bulls Thiol-type thiamine derivatives pounds in the semen 0 l 2 3 4 dlluter (percent) 7 Control 0 80+++ 75+++ 70+++ A Thiamine propyl disulfide hydrochloride 0. 00625 80+++ 80+++ 70+++ 60+++ Thlamine tetrahydrofurturyl disulfide hydrochloride 0. 00625 s0+++ s0+++ 5+ 5+ 5+ 50+++-++ 35+++-++ Control 0 85+++ 80+++ 80+++ 70+++ B Thiamine propyl disulfide hydrochloride 0. 00625 85+++ 80+++ 75+++ 70+++ Thiamine tetrahydrofurinryl disulfide hydro- 0. 00625 chloride. 5+ 5+ 10+ 5+ 27 Example 14 Sheep semen collected from 2 bulls is diluted five times with an egg yolk citrate buflier solution (the first diluter) containing 2.4% of sodium citrate, 20% of fresh yolk and the resulting diluted semen is cooled to 4 C. gradually for 1 hour. To the diluted semen is added egg yolk citrate buffer solution (The second diluter) containing 2.4% of sodium citrate, 20% of fresh yolk, 14% of glycerin and 0.0125% of thiamine propyl disulfide hydrochloride or thiamine tetrahydrofurfuryl disulfide hydrochloride or thiamine tetrahydrofurfury disulfide hydrochloride. After being kept standing at 4 C. for 12 hours, 1 ml. each of the diluted semen samples is put into a plastic straw of 1 ml. capacity and then the straws are closed. After being frozen and stored in a Dry Ice refrigerator at 79 C. for 7 days, each of the semen samples in the straws is thawed at 4 C. and stored at even temperature for 12 hours. The livability and motility of the respective thawed semen samples is shown in the following Table 14.

28 Example 16 taining 2.4% of sodium citrate, of fresh yolk, 14%

of glycerin and 0.0125 of thiamine tetrahydrofurfuryl disulfide hydrochloride at 4 C. The diluted semen is kept standing at 4 C. for 10 hours. 1 ml. each of the diluted semen samples is put into an ampoule of 2 ml.

15 capacity and the ampoules are closed. After being frozen and stored in a liquid-nitrogen refrigerator at 180 C. for 1 to 2 weeks, each of the ampouled semen preparations is thawed at 4 C., and subsequently inseminated to the female animals. The results are shown in the follow- TABLE 14 Concentrat tion of h es com- 12 outs Before Just after Bulls Tinoi-type thiamine derivative pounds in n the second freezing thawing thawing fluter (percent) Control 0 0s+++ 1o++ 4 A Thiamine propyl disulfide hydrochloride 0.0125 80+++ 10+ 10+ Thiamine tetrehydroiuriuryl disulfide hydro- 0 0125 80+++ chloride. 10+ 5+ Control 0 so+++ 20++-+ 75 B Thiamine propyl disulfide hydrochloride 0. 0125 85+++ 7 45+ Thiamine tetrahydrofurfuryl disulfide hydro- 0. 0125 85+++ chloride. 10+ 10+ Example 15 Fertility test is conducted on the bovine semen preparation of the present invention. Bovine semen collected from 4 bulls in diluted 10-fold with an egg yolk citrate ing Table 16. In the results, percentage of conception comes from the first insemination.

TABLE 16 buffer solution (containing 2.4% sodium citrate and 20% of egg yolk) supplemented with 0.0062 5% of thlamlne Controls (not containing propyl disulfide hydrochloride. The diluted semen 1s thiamine tetrahydrofurfuryl Samples inseminated to the female animals after being stored at Buns disulfide hydmchlmde) 4 C. for 1 to 5 days. The results are shown in the folb t b t Num ero Num BIO lowing Table 15. In the results, percentage of conception inseminated Percentage of inseminated Percentage 01 comes from the first insemination. animals conception animals conception TABLE 15 A 181 49.2 35 68.2 B 122 70.5 74 82.4 Controls (not containing C 86 147 thiamine pro yl disulfide Samples hydrochloride Bulls Number of Number of inseminated Percentage of inseminated Percentage 01 Having thus dlsclosed our mvent1on, what is claimed 1s. animals conception animals concep 1. A stabilized semen preparation for artificial insemination of livestock, which is composed of semen 1,;90 52% fig 238 of the livestock, a semen diluter and about 0.001% to 2-? 4 28 60 7 about 0.1% (weight/volume) of a thiol-type thiamine 1 57 1 16 62 5 derivative selected from the group consisting of unsymmetrical thiamine organic disulfide derivatives, S-acylthiamine derivatives, bisthiamine-disulfide derivatives and mixture thereof.

2. A stabilized semen preparation for artificial insemination of ruminant livestock, which is composed of semen of ruminant livestock, a semen diluter and about 0.001% to about 0.1% (weight/volume) of a thiol-type thiamine derivative selected from the group consisting of unsymmetrical thiamine organic disulfide derivatives, S- acyl-thiamine derivatives bisthiamine-disulfide derivatives and mixture thereof.

3. The stabilized semen preparation claimed in claim 2, wherein the ruminant livestock is selected from the group consisting of cattle, goat and sheep.

4. The stabilized semen preparation as claimed in claim 2, wherein the thiol-type thiamine derivative is an unsymmetrical thiamine organic disulfide derivative selected from the group consisting of thiamine propyl disulfide, thiamine tetrahydrofurfuryl disulfide, thiamine allyl disulfide, thiamine (7 methoxycarbonyl 3-acetylthioheptyl) disulfide, thiamine 2-hydroxyethyl disulfide and mixture thereof.

5. The stabilized semen preparation as claimed in claim 2, wherein the thiol-type thiamine derivative is a S acyl thiamine derivative selected from the group consisting of 0,8 diacetyl thiamine, O,S dibenzoylthiamine, S acetyl thiamine O monophosphate, O,S- dicarboethoxy thiamine, O,S-cyclocarbo thiamine and mixture thereof.

6. The stabilized semen preparation as claimed in claim 2, wherein the thiol-type thiamine derivative is a bisthiaminedisulfide derivative selected from the group consisting of thiaminedisulfide, O-benzoylthiamine-disulfide and mixture thereof.

7. The stabilized semen preparation as claimed in claim 2, wherein the stabilized semen preparation contains about 0.006% to about 0.01% (weight/volume) of the thiol-type thiamine derivative.

8. The stabilized semen preparation as claimed in claim 4, wherein the unsymmetrical thiamine organic disulfide derivative is thiamine tetrahydrofurfuryl disulfide.

9. The stabilized semen preparation as claimed in claim 4, wherein the unsymmetrical thiamine organic disulfide derivative is thiamine propyl disulfide.

10. The stabilized semen preparation as claimed in claim 2, wherein the semen diluter is selected from the group consisting of egg yolk diluter and milk diluter.

11. The stabilized semen preparation as claimed in claim 10, wherein the semen diluter is an egg yolk citrate buffer solution.

12. The stabilized semen preparation as claimed in claim 10, wherein the semen diluter is an egg yolk phosphate buffer solution.

13. The stabilized semen preparation as claimed in claim 10, wherein the semen diluter is a milk diluter which is prepared by heating whole milk or skim milk.

14. A semen diluter for artificial insemination of livestock, which is composed of a basal semen diluter and about 0.001% to about 0.1% (weight/volume) of a thioltype thiamine derivative selected from the group consisting of unsymmetrical thiamine organic disulfide derivative, S acyl thiamine derivatives, bisthiamine-disulfide derivatives and mixture thereof.

15. A semen diluter for artificial insemination of ruminant livestock, which is composed of a basal semen diluter and about 0.001% to about 0.1% (weight/ volume) of a thiol-type thiamine derivative selected from the group consisting of unsymmetrical thiamine organic disulfide derivatives, S acyl thiamine derivatives, bisthiamine disulfide derivatives and mixture thereof.

16. A semen diluter as claimed in claim 15, wherein the basal semen diluter is selected from the group consisting of egg yolk diluter and milk diluter.

17. A semen diluter as claimed in claim 15, wherein the basal semen diluter is an egg yolk citrate buffer solution.

18. A semen diluter as claimed in claim 15, wherein the basal semen diluter is an egg yolk phosphate buffer solution.

19. A semen diluter as claimed in claim 15, wherein the basal semen diluter is a milk diluter which is prepared by heating whole milk or skim milk.

20. The semen diluter as claimed in claim 15, wherein the thiol-type thiamine derivative is the unsymmetrical thiamine organic disulfide derivative selected from the group consisting of thiamine propyl disulfide, thiamine tetrahydrofurfuryl disulfide, thiamine allyl disulfide, thiamine (7 methoxycarbonyl 3 acetylthiaheptyl) disulfide, thiamine 2 hydroxyethyl disulfide and mixture thereof.

21. The semen diluter as claimed in claim 15, wherein the thiol-type thiamine derivative is a S acylthiamine derivative selected from the group consisting of 0,8 diacetyl thiamine, O,S dibenzoyl thiamine, S- acetyl thiamine O monophosphate, O,S dicarboethoxy thiamine, O,S-cyclocarbo thiamine and mixture thereof.

22. The semen diluter as claimed in claim 15, wherein the thiol-type thiamine derivative is a bisthamine-disulfide derivative selected from the group consisting of thiamine disulfide, O benzoylthiamine disulfide and mixture thereof.

23. The semen diluter as claimed in claim 15, wherein the semen diluter contains about 0.006% to about 0.01% (weight/volume) of the thiol-type thiamine derivative.

24. The semen diluter as claimed in claim 20, wherein the unsymmetrical thiamine organic disulfide is thiamine tetrahydrofurfuryl disulfide.

25. The semen diluter as claimed in claim 20 wherein the unsymmetrical thiamine organic disulfide is thiamine propyl disulfide.

26. A method for heightening and maintaining the livability and motility of spermatozoa in a semen preparation for artificial insemination of livestock, especially of ruminant livestock, which comprises incorporating a thiol-type thiamine derivative selected from the group consisting of unsymmetrical thiamine organic disulfide derivatives, S acyl thiamine derivatives, bisthiaminedisulfide derivatives and mixture thereof in the semen preparation in a concentration range from about 0.001% to about 0.1% (weight/volume).

27. The method according to claim 26, wherein the ruminant livestock is selected from the group consisting of cattle, goat and sheep.

28. The method according to claim 26, wherein the thiol-type thiamine derivative is an unsymmetrical thiamine organic disulfide derivative selected from the group consisting of thiamine propyl disulfide, thiamine tetrahydrofurfuryl disulfide, thiamine allyl disulfide, thiamine (7 methoxycarbonyl 3 acetylthioheptyl) disulfide, thiamine 2 hydroxyethyl disulfide and mixture thereof.

29. The method according to claim 26, wherein the thiol-type thiamine derivative is a S-acyl-thiamine derivative selected from the group consisting of O,S-diacetylthiamine, O,S-dibenzoyl-thiamine, S-acetyl-thiamine O- monophosphate, O,S-dicarboethoxy thiamine, O,S-cyclocarbo thiamine and mixture thereof.

30. The method according to claim 26, wherein the thiol-type thiamine derivative is a bisthiamine disulfide derivative selected from the group consisting of thiamine disulfide, O-benzoyl-thiamine-disulfide and mixture thereof.

31. The method according to claim 26, wherein the concentration range of the thiol-type thiamine derivative in the semen preparation is from about 0.006% to about 0.01% (weight/volume).

32. The method according to claim 28, wherein the unsymmetrical thiamine organic disulfide derivative is thiamine propyl disulfide.

References Cited UNITED STATES PATENTS 2,598,881 6/1952 Berliner 1951.8 3,005,756 10/1961 Van Demark et a1. 195l.8 3,016,380 1/ 1962 Yurugi et a1. 260256.5

32 Oto et a1. 260--256.5 Kawasaki et a1, 260--256.5 Smith et a1 1951.8 Folkers et a1. 195-18 S. R. ROSE, Primary Examiner US. Cl. X.R. 

